Cryogenically enhanced intravascular interventions

ABSTRACT

Techniques and devices for treating atherosclerotic disease use controlled cryogenic cooling, often in combination with angioplasty and/or stenting. A combination cryogenic/angioplasty catheter may cool the diseased blood vessel before, during, and/or after dilation. Controlled cooling of the vessel wall reduces actual/observed hyperplasia as compared to conventional uncooled angioplasty. Similar reductions in restenosis may be provided for other primary treatments of the blood vessel, including directional arthrectomy, rotational arthrectomy, laser angioplasty, stenting, and the like. Cooling of vessel wall tissues will often be performed through plaque, and the cooling process will preferably take the thermodynamic effects of the plaque into account.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application is a continuation application of U.S.patent application Ser. No. 09/511,191 filed on Feb. 23, 2000, which isa non-provisional of and claims the benefit of priority from U.S.Provisional Patent Application Serial No. 60/121,637 filed Feb. 24, 1999(Attorney Docket No. 18468-000500); and U.S. Provisional PatentApplication Serial No. 60/169,109 filed Dec. 6, 1999 (Attorney DocketNo. 18468-000510), the full disclosure of which are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to apparatus and methodsfor treating atherosclerotic disease. In a particular embodiment, thepresent invention provides a combination of controlled cryogenic coolingand balloon distention of a diseased vessel wall.

[0004] A number of percutaneous intravascular procedures have beendeveloped for treating atherosclerotic disease in a patient'svasculature. The most successful of these treatments is percutaneoustransluminal angioplasty (PTA). PTA employs a catheter having anexpansible distal end (usually in the form of an inflatable balloon) todilate a stenotic region in the vasculature to restore adequate bloodflow beyond the stenosis. Other procedures for opening stenotic regionsinclude directional arthrectomy, rotational arthrectomy, laserangioplasty, stenting, and the like. While these procedures have gainedwide acceptance (either alone or in combination, particularly PTA incombination with stenting), they continue to suffer from significantdisadvantages. A particularly common disadvantage with PTA and otherknown procedures for opening stenotic regions is the subsequentoccurrence of restenosis.

[0005] Restenosis refers to the re-narrowing of an artery following aninitially successful angioplasty or other primary treatment. Restenosistypically occurs within weeks or months of the primary procedure, andmay affect up to 50% of all angioplasty patients to some extent.Restenosis results at least in part from smooth muscle cellproliferation in response to the injury caused by the primary treatment.This cell proliferation is referred to as “hyperplasia.” Blood vesselsin which significant restenosis occurs will typically require furthertreatment.

[0006] A number of strategies have been proposed to treat hyperplasiaand reduce restenosis. Previously proposed strategies include prolongedballoon inflation, treatment of the blood vessel with a heated balloon,treatment of the blood vessel with radiation, the administration ofanti-thrombotic drugs following the primary treatment, stenting of theregion following the primary treatment, and the like. While theseproposals have enjoyed varying levels of success, no one of theseprocedures is proven to be entirely successful in avoiding alloccurrences of restenosis and hyperplasia.

[0007] It has recently been proposed to prevent or slow reclosure of alesion following angioplasty by remodeling the lesion using acombination of dilation and cryogenic cooling. Co-pending U.S. patentapplication Ser. No. 09/203,011, filed Dec. 1, 1998 (Attorney Docket No.18468-000110), the full disclosure of which is incorporated herein byreference, describes an exemplary structure and method for inhibitingrestenosis using a cryogenically cooled balloon. While these proposalsappear promising, the described structures and methods for carrying outendovascular cryogenic cooling would benefit from still furtherrefinements and improvements.

[0008] In light of the above, it would be desirable to provide improveddevices, system, and methods for treatment of diseased blood vessels. Itwould be further desirable if these improved techniques were compatiblewith known methods for treating atherosclerotic disease, but reduced theactual occurrence and/or extent of restenosis due to hyperplasia. Itwould be particularly desirable if these improved techniques werecapable of delivering treatment in a very safe and controlled manner soas to avoid injury to adjacent tissues. These devices, systems, andmethods should ideally also inhibit hyperplasia and/or neoplasia in thetarget tissue with minimum side effects, and without requiring a complexcontrol system or making a physician introduce numerous differenttreatment structures into the target area. At least some of theseobjections will be met by the invention described hereinafter.

[0009] 2. Description of the Background Art

[0010] A cryoplasty device and method are described in WO 98/38934.Balloon catheters for intravascular cooling or heating of a patient aredescribed in U.S. Pat. No. 5,486,208 and WO 91/05528. A cryosurgicalprobe with an inflatable bladder for performing intrauterine ablation isdescribed in U.S. Pat. No. 5,501,681. Cryosurgical probes relying onJoule-Thomson cooling are described in U.S. Pat. Nos. 5,275,595;5,190,539; 5,147,355; 5,078,713; and 3,901,241. Catheters with heatedballoons for post-angioplasty and other treatments are described in U.S.Pat. Nos. 5,196,024; 5,191,883; 5,151,100; 5,106,360; 5,092,841;5,041,089; 5,019,075; and 4,754,752. Cryogenic fluid sources aredescribed in U.S. Pat. Nos. 5,644,502; 5,617,739; and 4,336,691. Thefollowing U.S. Patents may also be relevant to the present invention:U.S. Pat. Nos. 5,458,612; 5,545,195; 5,733,280; 5,902,299; and5,868,735. The full disclosures of each of the above U.S. patents areincorporated by reference.

SUMMARY OF THE INVENTION

[0011] The present invention provides new techniques for treatingatherosclerotic disease using controlled cryogenic cooling. Theinvention may make use of a combination cryogenic/angioplasty catheter,eliminating any need for an exchange procedure to be preformed betweendilation of a stenotic region within a vessel wall and the applicationof cryogenic cooling to inhibit hyperplasia. The cooling catheter may besuitable for cooling the diseased blood vessel before, during, and/orafter dilation. Advantageously, controlled cooling of the vessel wallchanges its mechanical properties so as to enhance the ease ofconcurrent and/or subsequent dilation. More specifically, the coolingprocess may weaken the vessel and allows it to be expanded with a muchlower balloon pressure than with conventional uncooled angioplasty.Controlled cooling of the vessel wall has been found to effectivelyreduce actual and/or observed hyperplasia as compared to conventionaluncooled treatment of the blood vessel. Reductions in restenosis may beprovided for primary treatments of the blood vessel includingangioplasty, directional arthrectomy, rotational arthrectomy, laserangioplasty, stenting, and the like. Cooling of the vessel wall willoften be performed through plaque, and the cooling process willpreferably take the thermodynamic effects of the plaque into account soas to enhance efficacy while inhibiting morbidity.

[0012] In a first aspect, the present invention provides a method fortreating hyperplasia or neoplasia of a blood vessel region. The methodcomprises cooling an inner surface of the blood vessel region to atemperature and for a time sufficient to remodel the blood vessel suchthat observed subsequent excessive cell growth-induced stenosis of theblood vessel is reduced as compared to a stenosis of an equivalentlytreated uncooled blood vessel region.

[0013] Typically, the cooling will reduce stenosis by a relative amountof at least about 5% of the stenosis which would otherwise occur in thevessel, preferably by at least about 10%, and more preferably by atleast about 25%. Ideally, the cooling step effects a relative reductionof the stenosis of at least about 50% of the equivalent vessel regionstenosis, and may even be tailored to reduce stenosis by about 80% ormore. Measured reductions in absolute stenosis percentages often measuremore than 6%, preferably being more than 8%, and in experimentsdescribed herein, have been shown to be more than 15% and even betterthan 22%. Such benefits are provided by cooling times in a range fromabout 10 to about 30 seconds, and with the cooling temperature of theinner surface of the blood vessel being in a range from about 4° C. toabout −31° C. (preferably being in a range from about −5° C. to about−15° C.).

[0014] In another aspect, the invention provides a method for inhibitingrestenosis of a blood vessel region of a mammal. The blood vessel regionis subjected to a primary treatment effecting an initial reduction instenosis and inducing the restenosis. Typical primary treatments includedirectional angioplasty, arthrectomy, rotational arthrectomy, laserangioplasty, stenting, and the like. The method comprises cooling aninner surface of the blood vessel region to a temperature and for a timesufficient to remodel the blood vessel region such that observedrestenosis of the blood vessel is measurably inhibited. Typically, thecooling step induces at least one of apoptosis, cell membrane damage,and programmed cell death so as to provide these advantages.

[0015] In another aspect, the invention provides a method for inhibitingrestenosis of a blood vessel region. The blood vessel region issubjected to a primary treatment effecting an initial reduction instenosis and inducing the restenosis. The method comprises cooling aninner surface of the blood vessel region, and then reducing cooling sothat the inner surface of the blood vessel warms. The warmed innersurface is re-cooled so as to define at least onecooling/warming/cooling cycle. The at least one cycle has coolingtemperatures and cooling times sufficient to remodel the blood vesselregion such that the restenosis of the blood vessel is measurablyinhibited.

[0016] In another aspect, the invention provides a method for treating ablood vessel. The blood vessel has plaque disposed between a lumen and avessel wall of tissue. The method comprises cooling the vessel walltissue to a temperature sufficient to inhibit excessive subsequent cellgrowth-induced stenosis of the blood vessel. This cooling step isperformed by engaging a surface of the plaque with a cooling surface,and cooling the plaque with the cooling surface so that the plaque coolsthe vessel wall tissue.

[0017] Preferably, the vessel wall tissue will be cooled to a targettemperature in the range from about −4° C. to about −15° C. It should benoted that the cooling surface will often cool the lesion to atemperature significantly below that of the target temperature, as asignificant thermogradient may exist between an inner surface of theplaque and a plaque/endothelial tissue interface. In fact, the coolingsurface may cool the plaque to a temperature below the −5° C. to −15° C.range.

[0018] In many embodiments, the vessel wall may be cooled to the targettemperature for less than about 20 seconds, typically being cooled forat lest about 10 seconds. A rate of change of temperature of the vesselwall tissue may be significantly less than a rate of change of a plaquesurface temperature, again in recognition of the thermodynamic effectsof the plaque. Hence, in part because the presence of the plaque, thevessel wall tissue may stay at a reduced temperature for a significantamount of time after cooling is terminated. In general, at least one ofthe characteristics of the cooling process, such as a temperature of thecooling surface and/or a cooling time, may be determined at least inpart based on a thickness of the plaque, as the plaque may have asurprisingly large impact on the cooling regimen to provide the desiredtissue temperature cycle.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 schematically illustrates a combined cryogenic/angioplastysystem including a catheter with an angioplasty balloon that is axiallydisplaced from a cryogenic balloon.

[0020]FIG. 2 illustrates an alternative distal end of thecryogenic/angioplasty catheter for use in the system of FIG. 1, in whicha cryogenic balloon is nested within an angioplasty balloon.

[0021]FIG. 3 is a cross-section taken along the catheter body of thecryogenic/angioplasty system of FIG. 1.

[0022]FIGS. 4A and 4B illustrate cryogenic cooling temperatures providedby expansion of N₂O.

[0023]FIGS. 5 and 5A illustrate a particularly preferred controlledtemperature cryogenic catheter in which a saline solution having apredetermined freezing temperature controls the cooling of tissues bythermally coupling an inexpansible heat exchanger with a surroundingangioplasty balloon.

[0024]FIGS. 6A through 6C schematically illustrate a method for usingthe controlled temperature cryogenic balloon of FIG. 5.

[0025]FIGS. 6D and 6E are partial cross-sections schematicallyillustrating methods for selectively cooling a tissue of the vessel wallto a desired temperature through a lesion along the vessel lumen such asplaque.

[0026]FIGS. 7 and 7A illustrate an alternative cryogenic treatmentcatheter.

[0027]FIGS. 8A through 12C are graphical results of experiments showingan actual and observed reduction in restenosis and hyperplasia asdescribed in the three Experimental sections provided hereinbelow.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0028] The devices, systems, and methods of the present invention arerelated to those of co-pending U.S. patent application Ser. No.09/203,011, filed on Dec. 1, 1998 for an Apparatus and Method forCryogenic Inhibition of Hyperplasia. That application is assigned to thepresent assignee, and its full disclosure is incorporated herein byreference.

[0029] Referring now to FIG. 1, an exemplary system 10 is capable oftreating a diseased vessel wall of a blood vessel using a combination ofboth angioplasty dilation and cryogenic cooling. In general, system 10includes a catheter 12 coupled to a cryogenic fluid supply system 14 andan angioplasty pressurization system 16. One or both of cryogenic system14 and pressurization system 16 may be operatively coupled to acontroller 18 for coordination of cooling and dilation, as will bedescribed in more detail hereinbelow.

[0030] Catheter 12 generally includes a catheter body 20 having aproximal end 22 and a distal end 24. A proximal housing 26 includes anumber of ports for coupling of cryogenic system 14, pressurizationsystem 16, and the like to the proximal end of the catheter body. Anangioplasty balloon 28 and a cryogenic balloon 30 are mounted near thedistal end 24 of catheter body 20. The catheter body will generally beflexible and contain a plurality of lumens to provide fluidcommunication between the ports of proximal housing 26 and balloons 28and 30.

[0031] Angioplasty balloon 28 may be formed from a variety of materialsconventionally used for dilating blood vessels. Angioplasty balloon 28will typically comprise a non-dispensable material such as polyethyleneterephthalate (PET). Such angioplasty balloons are formed in a varietyof sizes depending on their intended use, typically having a length in arange from about 15 mm to about 50 mm and an expanded diameter in arange from about 2 mm to about 10 mm. Prior to inflation, angioplastyballoon 28 will generally remain in a low profile configuration suitablefor insertion into and maneuvering through the vascular system. Aguidewire lumen 32 extends through angioplasty balloon 28 and cryogenicballoon 30 from a proximal guidewire port 34 to facilitate accessing thetarget treatment site.

[0032] Angioplasty balloon 28 is inflated by injecting fluid frompressurization system 16 into a pressurization lumen 36 through apressurization port 38. In the embodiment of FIG. 1, balloon 28 willpreferably be isolated from balloon 30, so as to avoid inadvertentinflation of the cryogenic balloon during dilation. High contrastmarkers may be provided within the balloon to enhance an image of thedistal end of the catheter and facilitate positioning of the balloonfluoroscopically, sonographically, or under any other alternative imagemodality (with appropriate contrast structures). Such markers may beformed by winding a gold or platinum wire around the tubular structuredefining pressurization lumen 36, as illustrated.

[0033] In the embodiment of FIG. 1, cryogenic balloon 30 is disposedproximally of angioplasty balloon 28. This arrangement is advantageousfor first at least partially dilating the vessel wall and then treatingthe dilated vessel wall with cryogenic cooling, which can facilitatepositioning of the cryogenic balloon within an occluded region of thevessel. In alternative embodiments, the cryogenic balloon may bedisposed distally of the angioplasty balloon.

[0034] The structure and operation of cryogenic balloon 30 may beunderstood with reference to FIGS. 1 and 2, and also with reference toU.S. patent application Ser. No. 09/203,011, previously incorporatedherein by reference. Cryogenic fluid will often be injected into acryogenic supply port 42 and passed toward cryogenic balloon 30 throughcryogenic supply lumen 44 within catheter body 20. The cryogenic fluidmay comprise cryogenic liquids or liquid/gas mixtures, optionallyincluding carbon dioxide, nitrous oxide, liquid nitrogen, or the like.As the cryogenic liquid passes from supply lumen 44 and into cryogenicballoon 30, it is preferably distributed both radially and axially by adiffuser 46. Diffuser 46 will generally comprise a tubular structurewith radially oriented openings. As the openings are radially oriented,diffuser 46 will direct the cooling fluid roughly perpendicularlyagainst the wall of cryogenic balloon 30, so that the heat transfercoefficient between the cooling vapor and balloon wall is quite even andquite high. This helps to reduce the temperature of the balloon wall andprovides greater heat extraction for a given flow rate of coolant intothe balloon. Additionally, as the ports of diffuser 46 are distributedboth circumferentially and axially along the balloon, the diffuser canprovide a substantially uniform cooling over a significant portion of(often over the majority of) the surface of the balloon.

[0035] In some embodiments, the cryogenic cooling fluid may pass througha Joule-Thomson orifice between fluid supply lumen 44 and balloon 30. Inother embodiments, at least a portion of the cryogenic cooling fluid mayexit one or more ports into the balloon as a liquid. The liquid willvaporize within the balloon, and the enthalpy of vaporization can helpcool the surrounding vessel wall. The liquid may coat at least a portionof the balloon wall so as to enhance even cooling over at least aportion of the vessel wall. Hence, the ports of diffuser 46 may have atotal cross-section which is smaller than a cross-section of the fluidsupply of lumen 44, or which is at least as large as (or larger than)the cross-section of the fluid supply lumen.

[0036] After the cryogenic cooling fluid vaporizes within balloon 30, itescapes the balloon proximally along an exhaust lumen 48 and isexhausted from catheter 12 through an exhaust port 50. Inflation ofcryogenic balloon 30 may be controlled by the amount of cryogenic fluidinjected into the balloon, and/or by the pressure head loss experienceby the exhaust gases. Cooling is generally enhanced by minimizing thepressure within balloon 30. To take advantage of this effect so as tocontrol the amount of cooling a fixed or variable orifice may beprovided at exhaust port 50. Alternatively, a vacuum may be applied tothe exhaust port to control cooling and enhance cooling efficiency.

[0037] An exemplary structure for diffuser 46 may comprise a polyimidetube having an inner diameter of about 0.032 inches and a wall thicknessof 0.001 inch. Each port will define a diameter of about 0.0025 inches.There will typically be between six and six hundred ports in diffuser46. In the exemplary embodiment, four axial rows of ports are separatedby about 90° from each other. The rows are axially staggered so that theports in a single row have central line separations of about 4 mm, whilethe ports of adjacent rows are separated by about 2 mm. The overalllength of the porous diffuser tube will vary with the length of theballoon, and will typically be about 2 cm.

[0038] Diffuser 46 may be bonded concentrically about a central shaftdefining guidewire lumen 32. Adhesives seal the proximal and distal endsof the diffuser, or the diffuser can be incorporated at the distal endof tube 64 with an adhesive seal at the distal end of the diffuser. Highcontrast markers may again be provided to enhance an image of thecatheter and facilitate positioning of cryogenic balloon 18 at thetreatment site. The cryogenic cooling fluid will generally be introducedthrough the annular space between the diffuser tube and the centralshaft proximally of the balloon. The central shaft will typicallycomprise a polyimide tube, but may alternatively include any of a widevariety of materials.

[0039] In some embodiments, a temperature sensor may be thermallycoupled to balloon 30 to monitor and/or control cryogenic cooling of thearterial wall. Temperature sensor 52 may optionally be disposed on aninner or outer surface of balloon 30, and is coupled to controller 18 bythermocouple leads 54. Temperature sensor 52 may comprise athermocouple, thermistor, or the like.

[0040] To inhibit restenosis, controller 18 will generally initiate,monitor, and/or control cooling of the tissue. Cryogenic supply 14 willoften inject sufficient cryogenic cooling fluid to effect a cooling rateof the tissue in a range from about 2° C. to about 30° C. per second. Inan exemplary treatment, the system will maintain the temperature in arange from about 0° C. to about −80° C., optionally at a temperature inrange from −5° C. to about −40° C., for a time between about 1 and 60seconds, ideally maintaining the tissue at a temperature in a range fromabout −5° C. to about −15° C. for a time from about 10 to about 20seconds. The efficacy of the therapy at inhibiting restenosis may beenhanced by repeatedly cooling the tissue to such temperatures forbetween 1 and 5 cooling cycles, typically repeating between 1 and 6cooling cycles every 60 seconds. Typical treatment cycles may cool asurface temperature of the endothelium down to about −10° C., then allowthe surface temperature to warm to about 0° C. Five of these coolingcycles might be performed in about 40 seconds. To provide cooling, acryogenic liquid or liquid/gas mixture comprising carbon dioxide,nitrous oxide, or the like may flow through the balloon at a rate in anaverage from about 100 to about 800 mg per second. Such cooling (andoptional cooling cycles) may induce apoptosis and/or programmed celldeath.

[0041] To accurately control and/or monitor the pressure withincryogenic balloon 30, proximal housing 26 may include a cooling balloonpressure monitoring port 56. The pressure monitoring port will be influid communication with the cryogenic balloon 30, preferably through adedicated pressure monitoring lumen (not shown). Signals from pressuremonitoring port 56 and a thermal couple connector 58 may be transmittedto the controller 18. This allows the use of a feedback control systemfor initiating, regulating, and halting the supply of cryogenic fluidfrom fluid supply system 14. More specifically, the controller willoften provide a control signal to the fluid supply system in response tosignals from pressure monitoring port 56 and/or thermal couple connector58.

[0042] Referring now to FIG. 2, an alternative combinationcryogenic/angioplasty catheter again includes both a cryogenic balloon30 and an angioplasty balloon 28. In this embodiment, cryogenic balloon30 is nested within angioplasty balloon 28, so that if the low pressurecooling balloon were to break during the procedure, the higher pressurecapability of the surrounding angioplasty balloon 28 would contain theexhaust gases until the flow of coolant was stopped. In other respects,the structure of this nested embodiment is quite similar to thatdescribed above.

[0043] In use, the nested cryogenic/angioplasty balloon catheter of FIG.2 may allow pre-cooling of a diseased vessel wall prior to dilation,cooling of a vessel wall after dilation, interspersed cooling/dilation,and even concurrent dilation during cooling. Advantageously, thecatheter need not be repositioned between the application of dilationpressure and cryogenic cooling. Hence, this nested embodimentfacilitates the immediate sequential pre- and/or post-cooling of thestenosed vessel wall, thereby giving a wide flexibility in the treatmentprotocol. Advantageously, the interaction of cooling and dilation may beprecisely prescribed and effected by controller 18 (see FIG. 1) withouthaving to wait for the surgeon to reposition the catheter.

[0044] Where the vessel wall is to be at least partially dilated priorto cryogenic cooling, angioplasty balloon 28 may be inflated first withcontrast liquid 40 (as used in conventional angioplasty). The contrastliquid may then be at least partially evacuated, allowing coolingballoon 30 to be inflated at a pressure that is lower than theangioplasty distention pressure. Inflation of cryogenic balloon 30pushes the angioplasty balloon against the diseased wall of the vessel,so that the cryogenic fluid 60 within the cryogenic balloon is thermallycoupled to the diseased vessel wall by both the cryogenic balloon walland the angioplasty balloon wall in series. To enhance heat flow throughthe balloon walls, a heat transfer enhancing material may be included incryogenic balloon 30 and/or angioplasty balloon 28, particularly wheretreatment temperatures of about −50° C. and below are desired. Forexample, the addition of between about 1% and 10% boron nitride in apolyethylene or other balloon polymer can significantly improve heattransfer of the entire system. Surprisingly, a significant temperaturedifferential may be found between an inner and outer surface of eachballoon during cooling. Hence, improving the thermal conductivity ofeach balloon wall disposed between cryogenic fluid 60 and the targetedwall of the vessel may provide significant benefits when cooling to lowtemperatures.

[0045] In alternative methods for using the nested cryogenic/angioplastyballoon of FIG. 2, cooling may be initiated prior to complete dilationof the stenosed region of the vessel. The cooling process may weaken themechanical properties of the vessel and allow it to be expanded ordilated at a much lower pressure than is used with conventionalangioplasty. For example, dilation of a cryogenically cooled vessel mayrequire inflation of angioplasty balloon 28 with a fluid pressure ofabout 2 bar, as compared to about 10 bar for conventional uncooledangioplasty on the same vessel wall. Simultaneous cryogenic cooling andangioplasty may reduce and/or eliminate medial vessel fractures, therebyinhibiting proliferative response after angioplasty. It should be notedthat at least some of these advantages may be provided by using a singleballoon coupled to both a cryogenic supply system 14 and apressurization system 16. Such a cooling/angioplasty catheter used inthis fashion may allow the operator to perform both angioplasty andcryogenic antiproliferative treatments with a single inflation cycle ofthe balloon.

[0046] Still further alternative treatment cycles are possible,including inflating a balloon with a room temperature gas at normalangioplasty pressures to dilate the vessel, and then inflating theballoon with a cryogenic fluid or other coolant to treat the dilatedarea so as to inhibit hyperplasia. Alternatively, a balloon may beinflated with a standard angioplasty contrast liquid at normalangioplasty pressures to dilate the vessel. The balloon may then beflushed with saline, and then flushed with a dry room temperature gas todry the cooling fluid path. After the cryogenic fluid path is dry, theballoon may be inflated with a coolant to treat the dilated area.Cooling cycles before angioplasty and/or before stenting may alsoprovide the antiproliferative response described above.

[0047] Referring now to FIG. 3, the structure of catheter body 20 isillustrated in cross-section. An outer sheath 62 partially definesexhaust lumen 48, the exhaust lumen here comprising an annular spacedisposed between the sheath and an inner jacket 64. In, the exemplaryembodiment, sheath 62 comprises a polyethylene tube having an innerdiameter of 0.058 inches and a wall thickness of about 0.003 inches. Theexemplary jacket 64 comprises a polyimide having an inner diameter of0.035 inches and a wall thickness of 0.001 inches.

[0048] Within jacket 64, a core shaft 66 defines guidewire lumen 32,while a cooling inlet tube 68 and an angioplasty pressurization tube 70define supply lumen 44 and pressurization lumen 36, respectively. Theexemplary cooling inlet tube comprises a polyester or polyimide, whilethe pressurization tube in the exemplary system may comprise a polyesteror high density polyester. A wide variety of alternative materials mightalso be used. Thermocouple leads 54 are insulated in a conventionalmanner.

[0049] The above discussion describes structures and techniques forenhancing the efficiency of cryogenic cooling within a blood vessel.However, cryogenic cooling is capable of inducing temperatures wellbelow the preferred antiproliferative treatment ranges of the presentinvention (typically in a range from about −5° C. to about −15° C.).Referring now to FIGS. 4A and B, expansion of N20 from an initialpressure of 500 psi and an initial temperature of 0° C. to finalpressures in a range from atmospheric pressure to 100 psi results in acryogenic cooling fluid temperature significantly colder than ourpreferred target tissue temperatures. As it may be convenient to makeuse of commercially available 500 psi N20, it may be beneficial toinclude an additional temperature control mechanism to provide ourdesired treatment temperatures. While it may be possible to maintainexpanded cryogenic fluid pressures above 100 psi, the use of such highpressure gases within the vasculature may involve a significant risk ofserious injury if the high pressure gases escape the catheter and enterthe blood stream.

[0050] Referring once again to FIG. 2, one simple technique for reducingtissue cooling without increasing fluid within our balloon structures isto add a layer of insulating material 72 between the cryogenic coolingfluid and the tissue engaging surface of the balloon. A suitableinsulation material might include a thin layer of expanded Teflon™(ePTFE) on an inner or outer surface of cryogenic balloon 30, on aninner or outer surface of angioplasty balloon 28, or the like. The ePTFElayer may have a thickness in the range from about 0.00025 inches toabout 0.001 inches. A wide variety of alternative insulation materialsmight also be used. Alternative active temperature control techniquesmight be used with or without such an insulation layer, including theuse of controller 18 as shown in FIG. 1.

[0051] A particularly advantageous temperature control mechanism can beunderstood with reference to FIGS. 5 and 5A. In this embodiment, acontrolled temperature cryogenic balloon catheter 80 again includes anangioplasty balloon 28, which here contains an inexpansible heatexchanger 82. Cooling of fluid inlet tube 68 releases the cryogeniccooling fluid within heat exchanger 82, but does not expand the heatexchanger into direct thermal contact with the balloon wall ofangioplasty balloon 28. Instead, a saline solution 84 thermally couplesthe heat exchanger to the outer surface of angioplasty balloon 28.

[0052] Saline solution 84 will generally have a predetermined freezingtemperature, and sufficient cryogenic cooling fluid will generally beprovided to heat exchanger 82 so that the saline solution is onlypartially frozen. As a result, the temperature of the saline solutionwithin angioplasty balloon 28 will be maintained accurately at thefreezing temperature. As the freezing temperature of saline may bevaried by changing the salinity, this provides a convenient controlmechanism to vary the treatment temperature. Specifically, a 6% salinesolution will freeze at about −3.5° C., while an 18% saline solutionwill freeze at about −14° C. By varying the salinity between about 6%and 18%, the temperature of the saline solution during cryogenic cooling(while the saline is partially frozen) can be selected within a rangefrom about 5° C. to about 15° C. In an exemplary embodiment, a 12%saline solution will provide a freezing temperature of about 8° C.,which is particularly advantageous for use with thecontrolled-temperature cryogenic catheter 80 illustrated in FIG. 5.

[0053] It should be understood that there may be a significanttemperature difference between the freezing temperature of salinesolution 84 and the surface temperature of the endothelium, and thatthis variation may depend on the particular angioplasty balloonstructure used. Nonetheless, these controlled temperature cryogeniccatheters can take advantage of the latent heat of freezing to providean accurate treatment temperature despite minor variations in thecryogenic cooling flow, exhaust gas pressure due to bending of thecatheter shaft, or the like.

[0054] It should be understood that a variety of fluids, and possiblyeven solids, might be used in place of saline solution 84. In general,temperature control will be provided where a thermally couplingstructure undergoes a change in phase involving a significant latentphase change energy. Nonetheless, saline is particularly preferred asits range of freezing temperatures can be easily controlled within thedesired range, and as it poses little risk in the event of releasewithin the vasculature. Optionally, contrast may be included with thesaline solution to improve imaging of the system within the patientbody.

[0055] In the exemplary embodiment illustrated in FIGS. 5 and 5A, heatexchanger 82 extends proximally from angioplasty balloon 28 to at leastin part define exhaust lumen 48. This simple proximal tubular structure(which is referred to herein as an evaporator 84) may comprise apolyimide tube having an inner diameter in a range from about 0.036inches to about 0.051 inches, ideally having an inner diameter of about0.045 inches. Cooling inlet tube 68 within evaporator 84 and heatexchanger 82 may comprise a polyimide tube having an inner diameter in arange from about 0.005 inches to about 0.012 inches, ideally having aninner diameter of about 0.009 inches.

[0056] It is possible that evaporator 84 extending the entire length ofthe catheter may cause sufficient cooling of the saline proximally ofthe angioplasty balloon to induce freezing along catheter body 20. Toreduce this, an insulation jacket may be provided around the evaporatorproximally of heat exchanger 82. The insulation jacket may comprise apolyimide tube, preferably leaving a gap (as small as 0.001 inches)between the insulation jacket wall and evaporator 84. Alternatively, theballoon inflation lumen may be altered to prevent the saline fromthermally coupling exhaust lumen 48 to outer sheath 64. For example, apolyimide tube having an inner diameter in a range from about 0.012inches to about 0.025 inches may be disposed between evaporator 84 andouter sheath 62, with this additional tubular structure providing fluidcommunication between inflation port 38 and angioplasty balloon 28. Ineither of these embodiments, an additional port may be provided on theproximal housing in communication with the insulation gap (eitherbetween the insulation jacket and evaporator 84 or between evaporator 84and outer sheath 62) such that at least some of the air could beevacuated from this gap to reduce heat transfer to in the bloodsurrounding catheter body 20 and the exhaust gases.

[0057] A method for using controlled-temperature cryogenic catheter 80is illustrated in FIG. 6A through C. Typically, the catheter isintroduced into the vasculature through an introducer sheath, most oftenusing the widely known Seldinger technique. A guide wire GW ismaneuvered through the vessel, and catheter 80 is advanced over theguide wire and positioned adjacent diseased portion DP of vessel wallVW.

[0058] Once angioplasty balloon 28 is in position, the balloon may beinflated in a conventional manner through inflation port 38 to dilatethe vessel, as illustrated in FIG. 6B. Optionally, the vessel may bedilated using conventional contrast fluid to facilitate fluoroscopicallydirecting dilation. When standard contrast has been used for dilation,the balloon may be evacuated and filled with a saline solution whichfreezes at the desired treatment temperature. Alternatively, dilationmay be performed using this saline solution to avoid any delay betweendilation and cryogenic treatment. In still further alternativetreatments, cryogenic cooling may be initiated prior to or duringdilation. Regardless, prior to cooling the saline solution having thepredetermined freezing temperature will preferably be used to inflateangioplasty balloon 28 with sufficient pressure to provide good contactbetween the balloon and vessel wall VW. Typically, the angioplastyballoon will be inflated by the saline solution to a pressure in a rangefrom about 5 psi to about 30 psi, as illustrated in FIG. 6B.

[0059] To initiate cooling, a cryogenic fluid (usually in the form of aliquefied refrigerant or liquid/gas mixture) is injected into cryogenicsupply port 42. The cryogenic fluid flows through fluid supply lumen 44and is transmitted into heat exchanger 82, where it rapidly absorbs heatand vaporizes, thereby cooling saline 84 and angioplasty balloon 28. Asthe coolant vaporizes, it passes proximally along evaporator 84 toexhaust port 50. Sufficient cryogenic cooling fluid is supplied topartially freeze saline 84, so that the saline liquid/solid mixtureremains at about freezing temperature. Optionally, additional cryogeniccooling fluid may be introduced, with the freezing of the salineproviding a temporary plateau along the temperature excursion profile.More typically, the partially frozen saline melts by absorbing heat fromthe surrounding body. In the meantime, the cooling of saline withinangioplasty balloon 28 results in treatment of a surface layer 88 ofvessel wall VW engaged by the angioplasty balloon to an accuratelycontrolled treatment temperature in a range from about −5° C. to about−15° C. As a result, this treated tissue layer undergoes apoptosis,thereby avoiding and/or reducing the proliferative response of theluminal wall to dilation.

[0060] As can be understood from the device descriptions provided above,proper design of a cooling catheter can help to provide accuratetreatment temperatures in a range from about −5° C. to about −15° C. Inmany embodiments, it will be desirable to maintain the target tissuewithin this range for a time between about 20 to about 60 seconds. Asdescribed in detail in the Experimental sections hereinbelow, accuratelyproviding such treatment temperatures and times for treatment of thetissue can result in apoptosis without excessive immediate necrosis ofthe tissues of the vessel wall. Referring now to FIGS. 6D and 6E, manyvessels targeted for a primary treatment such as angioplasty, stenting,atherectomy, and the like, may have a significant amount of plaque Pdisposed between a tissue of the vessel wall VW and the vessel lumen L.Plaque P can have a surprisingly large thermodynamic effect on thetreatment of the tissues of vessel wall VW, as a significant temperaturegradient may exist between a surface of the plaque adjacent lumen L anda plaque/vessel wall interface. Hence, providing a cooling surface suchas a cooled balloon 28 (or optionally, a cooled liquid surface, or thelike) engaging an inner surface of plaque P at a target temperature forcryotherapy may not reduce the temperature of the vessel wall tissuesufficiently to inhibit hyperplasia. Similarly, if the tissue of vesselwall VW is cooled accurately down to the desired treatment temperature,and if cooling is then maintained for the desired treatment time, theinsulating effect of the plaque may maintain the vessel wall tissue at areduced temperature for a significantly excessive amount of time.

[0061] To properly accommodate the conditions within a particularpatient P, a thickness T of plaque P may be measured using anintravascular ultrasound system 90 as schematically illustrated in FIG.6D. An exemplary ultrasound system is commercially available from Scimedof Maple Grove, Minn. Alternative methods for measuring plaque and otherlesions within a lumen of a vessel include angiography, computertomography, and a variety of other known medical sensing modalities.

[0062] Once thickness T of plaque P has been measured, a treatment timeand/or temperature for cooling an outer surface of balloon 28 can bedetermined. Treatment times and/or temperatures may be calculated usinga mathematical model which accounts for the insulating effect of plaqueP. Alternative methods for determining treatment parameters may be basedon dosimetry (based on prior measured treatments), or the like.Treatment parameters for maintaining cooling using balloon 28 may beadjusted for a thickness, thermal conductivity, or other characteristicof plaque P. For example, it may take approximately 10 seconds to coolthe tissue of vessel wall VW to a treatment temperature in a range fromabout −5° C. to about −15° C. due in part to the presence of plaque P.Additionally, natural warming of the tissues of vessel wall VW may beinhibited after active cooling of balloon 28 has ceased due to theinsulating effects of plaque P. In fact, active cooling for a time in arange from about 10 seconds to about 20 seconds after the vessel wall VWreaches the treatment temperature may be preferred, as the vessel walltissue can remain at or near the treatment temperature due to theinsulated, gradual cooling of the tissue. Even with this abbreviatedactive cooling of the balloon, the total cooling treatment may becomparable to a treatment in which active cooling of a balloon in directcontact with the vessel wall tissue is maintained for a time and a rangefrom about 20 to about 60 seconds after temperatures reach a targettemperature between about −5° C. and about −15° C.

[0063] In the following Experimental sections, treatment temperaturesand times are generally given for tissue in substantially direct contactwith the cooling surface. Where significant amount of plaque is presentwithin a lumen of the vessel wall, the thermodynamic effects of thatplaque will preferably be taken into account in the treatment cycle ofthe cryotherapy device so as to effect the described tissue treatments.

[0064] Experimental I

In Vitro Studies of Arterial Freezing Injury

[0065] Abstract

[0066] Objective: To investigate the effects of hypothermia and freezingon human arteries at the cellular level.

[0067] Methods: Cultured arterial endothelial cells and smooth musclecells are chilled or frozen under controlled thermal conditions.Consequences such as necrosis or apoptosis, as well as the impact onlong term reproductive viability are measured with a variety of assays.

[0068] Results: These data establish correlations between thermalconditions and the extent and nature of arterial freezing injury.

[0069] Introduction

[0070] The paradoxical ability of freezing to produce eitherpreservation or destruction of biological tissues is well documented byresearchers in cryobiology. The impact of cold exposure on biologicalsystems is determined by precise thermal conditions such as finaltemperature, cooling and warming rates, and exposure dine, as well asbiophysical parameters specific to each tissue type. In order to designa cryotreatment which will effectively assist in the prevention ofarterial restenosis it is desirable to understand the specificrelationships between thermal conditions and the degree and mechanismsof arterial freezing injury. With this objective, the followingdescribes in vitro studies of the effect of chilling and freezing on twomajor arterial constituents; endothelial cells and smooth muscle cells.

[0071] Materials and Methods

[0072] Materials: Human coronary artery endothelial cells (CAEC) andsmooth muscle cells (CASMC) obtained from Clonetics, are cultured inbasal media supplemented with 5% fetal bovine serum and Cloneticssupplied growth factors, and grown in a 37° C., humidified, 5% CO₂environment. Freezing or chilling of cellular suspensions is carried outusing a low temperature stage, which consists of a copper block machinedto allow the circulation of liquid nitrogen through the block. Athermocouple mounted to the stage provides feedback to a temperaturecontroller. This controller regulates the power input to athermoelectric heater fixed to the copper block, thereby holding thetemperature of the stage to within 1 degree Celsius of the desired,preset temperature.

[0073] Methods: Necrosis experiments: A 30 μl aliquot of cellularsuspension stained with trypan blue is pipetted onto a precooled glassmicroslide positioned on the freezing stage, and immediately coveredwith a cover slip. After the allotted exposure time, the microslide istransferred to a 37° C. surface to thaw. For a second freezing cycle,the slide is moved back to the cold stage for the desired period oftime, and then thawed on the 37° C. surface. Trypan blue is excluded byintact plasma membranes, so a count of stained vs. unstained cellsimmediately following the freezing treatment provides a measure of acutenecrosis as reflected by membrane integrity, in these experiments, cellsare subjected to final freezing temperatures ranging from +10 to −40° C.for 10, 20, and 60 second exposure times, and both single and doublefreeze/thaw cycles are considered.

[0074] Reproductive survival experiments: For each freezing condition, a125 μl aliquot of cellular suspension is placed in the well of asterile, precooled chamber slide on the low temperature stage. Thechamber slide is transferred to a 37° C. surface to thaw the suspension,and then moved back onto the freezing stage if a second freeze/thawcycle is desired. After thawing, the sample is transferred to a cultureflask in a sterile field at a seeding density of 1×10⁴ cells/cm², andincubated with growth medium mixed with 10% alamar blue. Alamar blue isan oxidation-reduction indicator, which yields a colorimetric change inresponse to metabolic activity. Using a plate reader, the reduction ofthe dye is monitored hourly for 4 hours after the freeze, and then at 24hour intervals for up to 6 days. Reduction of the dye provides aquantitative measure of the proliferation of the treated cells. Again, atemperature range of +10 to −40° C. is examined, and single and doublefreeze/thaw cycles are used with 60 second cold exposure times.

[0075] Apoptosis experiments: Freezing experiments are carried out withboth cellular suspensions and adherent cells. Cellular suspensions arefrozen on chamber slides on the low temperature stage as describedabove. Adherent cells are frozen or chilled by immersion of the cultureflasks in saline-ice baths which maintain the desired temperatures.After the allotted exposure time, the flasks are transferred to a 37° C.bath for 1 to 2 hours. The cells are removed from the flasks bytrypsinization, and the cellular DNA is extracted using a lysing kitpurchased from Boehringer-Mannheim. After elimination of RNA byincubation of the samples in RNase, the purified DNA is electrophoresedin a 1% agarose gel for 2.5 hours at 75 V. DNA is visualized withethidium bromide using a UV transilluminator. DNA fragmentationindicative of apoptosis is detected by separation of the DNA into acharacteristic ladder. In these studies, cell suspensions are exposed tofinal temperatures in the range of +5° to −15° C. for 60 seconds andundergo 1 to 4 freeze/thaw sequences. Adherent cells are cooled forlonger periods of time, experiencing temperatures of +5 to −15° C. for0.5, 1, 2, and 4 hours. Post freeze incubation times of 1 to 2 hours at37° C. are enforced to allow apoptosis to progress to the DNAfragmentation stage. To determine how the occurrence of cold shockapoptosis is dependent on the cell growth phase, 24, 48, 72, and 120hour cultures are used.

[0076] Results and Discussion

[0077] Necrosis experiments: The plots in FIGS. 8A through 8D presentnecrosis data obtained with trypan blue. These plots include data forcells exposed to final freezing temperatures for 60 seconds (circles),20 seconds (triangles), and 10 seconds, (squares). From these plots itis evident that cells suffer no acute membrane damage when chilled orfrozen to temperature above −5° C., and a substantial percentage ofcells survive freezing to temperatures as low as −15° C. A doublefreeze/thaw cycle increases cellular damage, however in the highertemperature range from +10 to −5° C. the majority of cells stillsurvive. The duration of cold exposure appears to have no effect on cellsurvival for the range of times considered in this study.

[0078] Reproductive survival experiments: The alamar blue reductioncurves shown in FIGS. 9A through 9H and FIGS. 10A through 10G, give ameasure of the number of metabolically active cells in each treatedculture, relative to a sham operated control. In FIGS. 9A through 9H,the percent reductions of alamar blue in CASMC cultures are plotted as afunction of time for sham operated control cells (diamonds), cellsexposed to a single freeze cycle (squares), and cells exposed to adouble freeze cycle (circles). In FIGS. 10A through 10G, percentreduction of alamar blue are plotted for CAEC cultures as a function oftime for sham operated control cells (diamonds), and for cells exposedto a single freeze cycle (squares). From these plots it is evident thatthe growth of cells exposed to temperatures in the range from +10 to−10° C. is nearly equivalent to the growth of untreated cells, for bothsingle and double freeze/thaw cycles over the course of 2-6 daysfollowing a cold treatment. At temperatures of −15 to −20° C. the growthof frozen cells is less than that of control cells, although some growthis still detected. At lower temperatures virtually no growth is found,and the cells are presumably 100% necrotic.

[0079] Apoptosis experiments: For the thermal conditions listed in themethods section, significant levels of cold shock induced apoptosis havenot been detected. Since the electrophoresis assay requires a minimumfraction of cells to be undergoing apoptotic DNA fragmentation for thecharacteristic DNA ladder to become visible, it is possible thatapoptotic events are occurring, but at a level too low to measure bythis assay. More sensitive assays such as the TUNEL method will be usedto quantify the effect of cold on cells in the higher non-necrotictemperature range, as described below.

[0080] Apoptosis

[0081] As discussed above, the goal of this study is to investigate theeffects of cold exposure on human arteries at the cellular level. Astudy of freeze induced necrosis has been described above. However,freezing injury may manifest itself in forms other than acute cellularnecrosis. An important mechanism of cellular death is apoptosis or“programmed cell death”, which is characterized by internucleosomalcleavage of DNA, cell membrane blebbing, condensation of nuclearchromatin, and the fragmentation of the cell into smaller apoptoticbodies.

[0082] Cold shock can induce apoptosis. In particular, apoptosis can betriggered by exposure to temperatures ranging from 0 to −15° C.,conditions which yield low levels of cellular necrosis. On this basis,studies were conducted to identify the role of apoptosis in the overallresponse to cold temperatures for the cells in this particular system.Apoptosis is especially interesting for this application because, unlikecellular necrosis, it does not cause the inflammation which marks theinitiation of neointimal hyperplasia. The studies described in thissection seek to assess whether the smooth muscle cells involved in theproliferative process of neointimal hyperplasia can be destroyed byinducing apoptosis rather than necrosis, thereby avoiding aninflammatory response. Because of the complexity of the apoptoticmechanisms, three assays have been applied to study the occurrence ofcold shock apoptosis: gel electrophoresis, a TUNEL assay, and an AnnexinV assay.

[0083] Experiments conducted with the gel electrophoresis assay weredescribed above. As reported there, significant levels of cold shockinduced apoptosis were not detected with the electrophoresis assay.However, this result does not necessarily rule out the presence ofapoptosis. The gel electrophoresis assay has certain limitations. Forinstance, the appearance of the characteristic ladder pattern isdependent on the presence of a minimum quantity of fragmented DNA,making the detection of a positive result dependent of the number ofcells in the sample population and the efficacy of the DNA isolationprocess, as well as the percentage of cells in the appropriate stage ofthe apoptotic process. Therefore, it is possible that apoptotic eventstook place, but at a level too low to measure by this assay. In additionto this, fragmentation of the DNA into characteristic lengths representsa specific stage in the apoptotic process. As that process continues,DNA fragments further and is sequestered in the smaller apoptotic bodieswhich are entirely digested by neighboring cells, so that thecharacteristic ladder is no longer produced. Thus, if the assay isapplied to a cell population too early (before fragmentation begins) ortoo late in the apoptotic process, it will fail to identify apoptosis.The time taken for a particular cell type to undergo each step of theapoptotic process in response to a particular driving mechanism isunknown and can vary from minutes to hours. It is possible thatsignificant levels of apoptosis were present in the treated samples, butthe cells were not lysed at the critical moment in the process. For thisreason the two other assays described below were used to evaluate theability to produce apoptosis in cells though cold exposure in thenon-necrotic temperature range.

[0084] Materials and Methods

[0085] Materials: Human coronary artery endothelial cells and ratarterial smooth muscle cells were used in these experiments, and weremaintained in culture as previously described. For these assays, thefreezing experiments were performed with adherent cell populations. Toprepare samples for experimentation., cells were seeded on 22 mmdiameter round, culture treated, Thermanox coverslips, which wereincubated with growth media in tissue culture plates.

[0086] Methods: TUNEL experiments: Like gel electrophoresis, the TUNEL(terminal deoxynucleotidyl transferase mediated dUTP nick end labeling)assay uses DNA fragmentation as the marker for apoptosis. This assay isbased on the principle that cleavage of genomic DNA during apoptosisyields single strand breaks (“nicks”), which can be identified bylabeling free 3′-OH termini with modified nucleotides in an enzymaticreaction. Incorporated fluorescein is detected by anti-fluoresceinantibody Far fragments from sheep, conjugated with horse-radishperoxidase (POD). After substrate reaction to produce a colorimetricprecipitate, stained cells can be analyzed under light microscopy.

[0087] The adherent samples were frozen by placing the coverslips onpre-cooled glass microslides positioned on the low temperature stage.Solidification was visually observed. To thaw, the coverslips wereremoved from the stage and dipped in growth medium at 37° C.Temperatures of +5 to −15° C., an exposure time of 60 seconds, and asingle freeze/thaw cycle were applied. After thawing, the cover slipswere returned to the culture plates, and incubated with growth media at37° C. Post freeze incubation times of 0, 0.5, 1, 2, 3, 4, and 24 hourswere tested, since the time required for apoptotic cells to reach theDNA fragmentation stage was unknown.

[0088] After the post-freeze incubation period, the adherent cells wereair dried, fixed in a 4% paraformaldehyde solution, and incubated with a0.3% H₂O₂ methanol solution to block endogenous peroxidase. Next, thecells were incubated in a permeabilisation solution to permitpenetration of the TUNEL enzyme in a subsequent step. Finally, the cellswere incubated in converter horse-radish peroxidase (POD) which binds toTUNEL labeled DNA strand breaks, and then in a DAB/metal enhancedsubstrate, which produced a dark brown precipitate in the presence ofbound POD. The coverslips were rinsed thoroughly with phosphate bufferedsaline between each of the incubations. After the final incubation inthe DAB substrate, the cover slips were analyzed under light microscopy.Hemotoxylin was applied as a counterstain in order to facilitate thecount of non-apoptotic cells. A positive control, included in eachexperiment., was prepared by incubating a fixed, permeabilized cellsample with DNase 1 to induce DNA strand breaks. Additionally, a shamoperated control sample which was exposed the same handling but not toany cold shock, was included in each experiment. In examining thesamples, care was taken to confirm that all positively stained cellsincluded in the count of apoptotic cells exhibited a morphologycharacteristic of apoptosis. These cells appeared shrunken or condensed,in contrast to the swollen, distorted appearance of necrotic cells.

[0089] Annexin V experiments: An early event in the process of apoptosisis the flipping or inversion of the molecules of the plasma membrane,causing phospholipid phosphatidylserine (PS) to be translocated from theinner leaflet of the membrane to the outer cell surface. The exposed PSserves as an identification tag utilized by Annexin V, a protein with ahigh affinity for phosphatidylserine. When apoptotic cells with exposedPS are incubated with the Annexin V compound, the Annexin binds to thePS on the membrane surface. The Annexin V bound sites are labeled with asecondary conjugate, and then prepared for analysis with a substratereaction that yields a colorimetric change visible to light microscopy.

[0090] The freezing of adherent cells on coverslips using the lowtemperature stage was performed as in the TUNEL experiments. Again,temperatures of +5 to −15° C., and a single freeze/thaw cycle wereapplied. The correlations established by the TUNEL data demonstratedthat the process of apoptosis is underway and has progressed to the DNAfragmentation stage between 1 to 2 hours after cold shock. Sincemembrane inversion is an earlier event in the apoptotic process, anincubation time of 1 hour was selected for this study. This timingallowed the membrane alterations to be detected before extensivedegradation of the cells into smaller bodies could occur. In the Annexinstudies, the correlation between cold exposure time and apoptosis wasalso investigated. Since only short exposure times were relevant to thisparticular application, cold exposure times of 30, 60, and 120 secondswere tested.

[0091] The Annexin assay was performed through the following series ofsteps. After the one hour incubation, the coverslips were removed fromthe culture plates and rinsed in phosphate buffered saline. They werethen incubated in the Annexin-V-Biotin working solution. Subsequently,the cells were air dried, fixed in a methanol/ethanol solution, airdried again, and then incubated with Streptavidin conjugated with horseradish peroxidase (POD). The Streptavidin labeled the bound Annexin, andthe POD provided a colorimetric reaction induced by exposure to aDAB/metal enhanced substrate. Following the substrate reaction, thecoverslips were mounted on slides and examined under light microscopy.

[0092] Since membrane integrity is lost in necrotic cells, the Annexin Vis able to penetrate into those cells, binding to the PS on the innerleaflet of the membrane, and positively staining the necrotic cells.Therefore simultaneous staining with trypan blue was required todifferentiate between necrotic and apoptotic cells. Note that althoughapoptotic cells undergo membrane inversion, the membrane remains intactand impermeant to exclusion dyes such as trypan blue. For eachexperimental condition, two samples were identically cold shocked. Oneof these samples was stained with the Annexin protocol as describedabove. The other sample was stained with trypan blue exclusion dye for 5minutes and then examined under light microscopy to establish thepercentage of necrotic cells induced by each thermal condition. Thispercentage was subtracted from the percentage of Annexin stained cells,to generate the percentage of apoptotic cells in each sample.

[0093] The plots in FIGS. 11A through 11F represent the findings ofthese assays. FIGS. 11A and 11B show the percentage of apoptotic cellsfound in samples of human coronary artery endothelial cells and ratarterial smooth muscle cells respectively, as a function of the finaltemperature to which they were frozen. TUNEL results are indicated witha dashed line, and Annexin results are indicated with a solid line. Aminimum of three experiments were conducted for each thermal condition,and the data points represent the averaged result. These plots show thatsignificant levels of apoptosis were found with both assays for thetemperature range examined here. The two cell types experience verysimilar levels of apoptosis in response to the cold shock. For both celltypes, apoptosis was not found at hyperthermic conditions (+5 to 0° C.),but was induced at lower temperatures, with a maximum response occurringat −10° C. A comparison between the Annexin results and TUNEL datareveals that the TUNEL assay produced somewhat higher levels of positivestaining. It should be noted that, whereas positive apoptosis resultsare difficult to obtain with the electrophoresis assay, the TUNEL assayhas a tendency to be biased towards positive outcomes. One possiblefactor contributing to falsely high apoptotic results is the positivestaining of necrotic cells. The TUNEL reaction preferentially labels DNAstrand breaks generated during apoptosis, however extensive DNAfragmentation may occur in late stages of necrosis leading to somepositive staining of necrotic cells. Although examination of themorphology of stained cells generally allowed discrimination betweennecrotic and apoptotic cells, in some fraction of the stained cells themorphological characteristics were inconclusive and the designation ofthe cells was uncertain.

[0094]FIG. 11C shows the relationship between TUNEL detected levels ofDNA fragmentation and post freeze incubation time for human coronaryartery endothelial cells. The cold exposure consisted of 60 second,single cycle freezes, and the results for three different finaltemperatures (−5, −10, and −15° C.) are presented in this plot. From thefigure it is evident that for each temperature, the maximum responsemeasured by the TUNEL assay was found within 1 to 2 hours after the coldshock. 24 hours after cold shock, little fragmentation was found. Thisindicates that DNA fragmentation reaches its peak at approximately 2hours after the apoptotic process begins.

[0095]FIGS. 11D and 11E show the relationship between the level ofapoptosis detected with the Annexin assay and the time of cold exposurefor human coronary artery endothelial cells and rat arterial smoothmuscle cells, respectively. In each plot, results for cold shock at −5and −10° C., in a single freeze cycle with a one hour post freezeincubation are presented. The figures reveal that the time of exposuredoes not significantly affect the percentage of apoptotic cells, withinthe 2 minute time range relevant to this application. The smallvariations in percentage of apoptosis are well within the range ofaccuracy of the Annexin assay.

[0096] In conclusion, the varied results of the three assays reflect thecomplex nature of the apoptotic phenomenon. Although the electrophoresisassay failed to positively identify a substantial apoptotic fraction,both the TUNEL and the Annexin assays demonstrated some contribution ofapoptosis in the overall response of arterial cells to cold shock. Thetwo assays consistently revealed an apoptotic peak at −10° C., and noapoptosis above −5° C. Notwithstanding the limitations in the assaysthemselves, exact percentages for apoptosis are difficult to establishbecause of the complexity of the mechanism. Any number of uncontrolledbiological factors, including the precise growth phase of the cells, andthe biological responsiveness and function of the cells, could affectthe initiation of the apoptotic process in an unknown way. This datashould therefore be viewed as evidence that significant levels ofapoptosis can be induced by cold exposure in the non-necrotictemperature range. Although the qualitative correlations betweentemperature, time, and peak apoptosis are reliable, the quantitativemeasurements should be considered approximations.

[0097] Experimental II

Cryogenic Intravascular Treatment for Inhibition of Neointimal Formationin the Balloon Injury Model

[0098] Objectives: The proliferative and morphometric response in theswine coronary model were compared after balloon injury to ballooninjury and intravascular cryogenic treatment.

[0099] In the rabbit carotid model, percent stenosis in cryogenicallytreated arteries was compared to sham arteries.

[0100] Methods: Seven pigs were treated with balloon overstretch(balloon to artery ratio 1.3:1) in at least two coronary arteries.Intravascular cryogenic treatment was administered in at least one ofthe arteries after overstretch using a cooled angioplasty balloon. BrdUwas administered and the animals were sacrificed on day 7.

[0101] Two rabbits (total eight sites in the carotid arteries) weretreated with a cooled or non-cooled angioplasty balloon. None of therabbit carotids received balloon overstretch. The rabbits weresacrificed at 28 days.

[0102] Results: In the swine, BrdU index in the sham measured 16% to 43%medial fracture 0% to 67.6%, and percent stenosis 0% to 36.6%. In thecryogenically treated arteries BrdU index measured 18% to 36%, medialfracture 0% to 44.5% and percent stenosis 1.9% to 16.9%.

[0103] In the rabbits, percent stenosis measured 3.5% to 10% in theshams and 3.1% to 7.4% in the cryogenically treated arteries. Medialfracture measured 0% in the shams and 0% to 2.4% in the cryogenicallytreated arteries.

[0104] Conclusion: Intravascular cryogenic treatment in the swine andrabbit vascular model effect cell proliferation and present stenosisfollowing a balloon injury.

[0105] Materials and Methods

[0106] Description of the Intravascular Cryogenic Treatment System: Theintravascular cryogenic treatment system includes a cryogenic ballooncatheter and a delivery system. The cryogenic balloon catheter (60-cmusable length) was mounted coaxially around a polyimide diffuser tube(0.034″ OD.×0.001″ wall) and a 0.020″ O.D. polyimide tube defining aguide wire lumen. A polyethylene balloon (4 cm in length) was mounted ona polyethylene catheter shaft (048″ I.D.×0.005′ wall). A manifold ismounted to the proximal end of the balloon shaft and provides ports forguide wire insertion, refrigerant inlet and refrigerant exhaust.

[0107] The polyimide diffuser tube connects the most distal end of theballoon to the manifold. The portion of the diffuser tube inside theballoon was fenestrated (40 holes, 0.0028″ dia. spaced evenly over 2 cm)to allow refrigerant to flow radially outward against the inside wail ofthe balloon. The polyimide refrigerant inlet tube (0.011″ O.D.×0.001″wall) resides in the coaxial space between the guide wire tube and thediffuser tube.

[0108] The refrigerant inlet tube, guide wire tube, balloon shaft anddiffuser tube are potted into the manifold using a medical gradeadhesive (Dymax 204-CTH). The refrigerant exhaust port, connects to anadjustable pressure relief valve and provides for control of thepressure inside the balloon. The valve is set between 60 and 120 psi.

[0109] The delivery system dispenses liquid nitrous oxide through theinlet port on the manifold. The nitrous oxide is contained in an 8-gramcylinder (675 psi at 23° C.) and is inverted so that the refrigerantflows through the inlet port of the manifold. A hand held housingsurrounding the liquid nitrous oxide cylinder incorporates a punctureneedle and seal on one end and a threaded plunger on the other end,enables the user to safely puncture the gas cylinder. A high-pressurestopcock containing a 20μ filter to trap particulates that may clog theinlet port, is mounted to the to the outlet of the delivery system.

[0110] Description of the Temperature Monitoring Guide Wire: A 0.014″coronary guide wire (ACS Extra S'port) was modified to measuretemperature at the vessel wall during the cryogenic treatment. Athermocouple (type ‘T’ and constructed from 0.0015″ bifilar wire coatedwith 0.0001″ polyurethane) was mounted 2 cm from the distal tip of theguide wire, covered with 1 cm of polyester (0.00025″ thick) and pottedwith adhesive to displace any air trapped adjacent to the thermocouple.

[0111] Operation of the Intravascular Cryogenic Treatment System: Afterthe balloon catheter and the thermocouple wire were positioned at thetreatment site, the nitrous oxide cylinder was punctured with thestopcock in the “off” position. The delivery system was then connectedto the inlet port on the manifold and the stopcock opened. The nitrousoxide passed through the inlet tube into the diffuser tube, sprayingradially outward against the inner surface of the balloon. The nitrousoxide evaporated in the balloon and was exhausted as a gas through thecoaxial space between the diffuser tube and the balloon shaft.

[0112] Swine Preparation and Treatment: Seven swine (35 to 50 kg)received Aspirin and Ticlid the day prior to the procedure and daily forseven days after the procedure. The animals were sedated and intubated.After placement of an 8F-introducer sheath in the left carotid artery bysurgical cut-down, each animal received a dose of heparin. An 8F CordisH-Stick guiding catheter was placed in the coronary ostium and a 0.014″coronary guide wire was advanced to the artery to be treated. Thecoronary arteries were imaged using a 2.9F or 3.2F (30 MHz)intravascular ultrasound catheter (IVUS) to identify appropriatetreatment sites (diameters ranging from 2.25 mm to 3.75 mm). Fifteenarteries were treated with balloon overstretch injury, 1.3:1 (3inflations for 30 sec., separated by 1 min. deflation periods to restorecoronary perfusion) following either cryogenic or sham treatment usingthe intravascular cryogenic treatment system with the temperaturemonitoring guide wire positioned between the balloon and the vesselwall. The sham arteries were treated using treatment catheters filledwith saline at pressures similar to cryogenic pressures used. Allanimals were recovered and survived for seven days. BrdU injections (50mg/kg) were administered twenty-four hours and one hour prior tosacrifice. Prior to euthanasia all animals were systemicallyheparinized. The coronary arteries were perfusion fixed at 75 mm Hg for20 minutes with 10% neutral buffered formalin. The hearts were removedand immersion fixed for at least 24 hours.

[0113] Rabbit Preparation and Treatment: Two Rabbits (4.5 and 5 kg)prepared in a similar fashion were treated with the intravascularcryogenic treatment system or sham catheter in the carotid artery. Bothfemoral arteries were accessed to accommodate the 4.5F treatmentcatheter (or sham catheter) and the temperature monitoring guide wire.The rabbits were survived and sacrificed on day 28. The carotid arterieswere perfusion fixed with 10% neutral buffered formalin. RESULTS TABLE I° C./Time % % Medical BrdU % Animal Animal # Artery (sec) StenosisFracture (neointimal) Swine  42 LAD −13/17 1.4 36.3 18 Swine  42 LCXsham 8.3% 27.5 43 Swine  24 LAD −31/24 10.6 44.5 24 Swine  24 LCX sham24.4 34.9 26 Swine  61 LCX −18/18 13.5 24.1 31 Swine  61 RCA sham 36.667.6 24 Swine 9001 LCX −28/24 1.9 0.0 not measured Swine 9001 LAD sham17.3 41.9 16 Swine 9002 LAD −21/30 9.36 38.1 36 Swine 9002 LCX sham 0.00.0 not measured Swine 9005 LCX −11/9 16.9 28.21 24 Swine 9005 LAD sham12.3 28.1 29 Swine 9006 LAD −12/6 4.6 30.6 31 Swine 9006 LCX sham 15.351.9 26 Rabbit 2997K Left-prox sham 10.0 0.0 N/A Rabbit 2997K Left-mid−15/23 7.4 2.4 N/A Rabbit 2997K Right-prox sham 9.2 0.0 N/A Rabbit 2997KRight-mid −21/25 5.4 0.0 N/A Rabbit 3131K Left-prox sham 5.7 0.0 N/ARabbit 3131K Left-mid −33/25 3.8 0.0 N/A Rabbit 3131K Right-prox sham3.5 0.0 N/A Rabbit 3131K Right-mid +4/0 3.1 0.0 N/A

[0114] Conclusions and Discussion

[0115] The rabbit data of Results Table I suggests that over a widerange of temperatures (+4 to −33° C.) the cryogenic treatment system didnot produce significant stenosis and in all cases produced less stenosiswhen compared to the sham in the same artery. The cryogenic treatment inthe rabbit carotids did no harm and healed in a benign fashion.

[0116] In the overstretch swine model of Results Table I, the moststriking result was observed in animal 42. The percent medial fracturewas highest in the cryogenically treated artery (36.6% in the treatedartery compared to 27.5% in the sham) however both the percent stenosisand BrdU index were lower. Overall, the morphometric analysis of treatedarteries verses sham arteries revealed a substantial reduction in thepercent stenosis as the cryogenic arteries averaged 8.3% verses 16.3% insham treated arteries.

[0117] Experimental III

Swine Coronary Pilot Trial

[0118] Purpose: To determine the vascular effects of a freezingtreatment in a stent overstretch swine coronary model.

[0119] Hypothesis: Endovascular cryothopathy applied to an injuredartery will result in similar or less neointimal formation than sham asjudged by histologic and morphometric examination at 28 days.

[0120] Animal Preparation: Four Yorkshire white pigs weighing 35 to 50kg were used for this study. On the day prior to the experiment, eachpig received Aspirin and ticlopidine. Following general anesthesia, theleft carotid artery of each animal was cleanly dissected and an 8 Fr.sheath was placed. Systemic anticoagulation was achieved withintravenous heparin.

[0121] After each experiment, the left carotid artery was repaired andthe animal was recovered from anesthesia. Animals were housed onstandard chow for 28 days following the experiments. For the duration ofthe recovery period the animals received Aspirin, and ticlopidine wasgiven for the first two weeks. On day 28, each animal was sacrificed inan ethical manner, and the heart was perfusion fixed at approximately 75mm Hg for 20 minutes in 10% buffered formalin. The arterial segments ofinterest were harvested and the tissue underwent routine histologic andmorphometric evaluation.

[0122] Device Description: The intravascular cryogenic treatment systemincluded a cryogenic balloon catheter, as shown in FIGS. 7 and 7A, and adelivery system. The cryogenic balloon catheter 110 (60 cm usablelength) was mounted coaxially around a polyimide fluid delivery tube 112(0.011″ I.D. by 0.001″ wall) and a 0.020″ O.D. polyethylene tubedefining a guide wire lumen 114. A polyethylene balloon 116 (3 cm inlength) was mounted on a polyethylene catheter shaft (0.165″ I.D. by0.005″ wall). A manifold 118 was mounted to the proximal end of theballoon shaft and provided ports for guide wire insertion, refrigerantinlet, refrigerator exhaust, and temperature measurement inside theballoon.

[0123] The guide wire tube connected the most distal end of the balloonto the manifold. The portion of the delivery tube inside the balloon,mounted coaxially around the guide wire lumen, allowed refrigerant toflow evenly against the inside wall of the balloon. The polyimiderefrigerant inlet tube 123 (0.011″ O.D. by 0.001″ wall) resided in thecoaxial space 119 between the guide wire tube 114 and balloon cathetershaft 110.

[0124] A “K” type thermocouple 120 constructed from a 0.0015″ by 0.003″bifilar wire was mounted inside the balloon. The thermocouple wire leads117 were positioned in the annular space between guide wire lumen 114and balloon catheter shaft 110. A connector 121 was mounted on manifold118.

[0125] The refrigerant inlet tube 123, guide wire tube 114, balloonshaft 110, and thermocouple wires 120 were ported into manifold 118using a medical grade adhesive (Dymax 204-CTH). The refrigerant exhaustport 122 connected to an adjustable pressure relief valve 124 andprovided for control of the pressure inside the balloon. The valve wasset at 15 psi.

[0126] The delivery system dispensed liquid AZ-50 126 (pentafluorothaneand trifluoroethane) through the inlet port on the manifold. The AZ-50was contained a 25 cc cylinder (330 psi at 23° C.) and was inverted sothat the refrigerant flowed through the inlet port of the manifold.

[0127] Experimental Methods: A guide catheter was advanced to the routeof the aorta and the coronary arteries were selectively engaged underfluoroscopic visualization. Each coronary artery was interrogated byIVUS to determine cross-sectional diameter. Arterial injury was inducedby percutaneous catheter implantation of intracoronary stents at astent-to-artery ratio of 1.0:1 in one animal (#9108) and 1.3:1 in theother three animals (Nos. 9195, 9196, and 9197). Following stentplacement, one artery in each animal underwent freezing treatment byinsertion of the cryogenic catheter (described above), with thetreatment zone centered in the stent. Refrigerant was delivered in tothe catheter for a period of 30 seconds, producing balloon inflation ata pressure of 15 psi, and a temperature of −10° C. at the balloon/arteryinterface. Cryogenic catheter sizes were chosen to provide goodapposition between the balloon and the artery wall. The other artery ineach animal was sham treated at the stent location using a ballooncatheter identical to the cryogenic catheter, but delivering notemperature change, inflated at 15 psi for 30 seconds.

[0128] Results: All animals underwent routine histologic evaluation. Theendpoint of the study lay in a comparison of the lumen area, neointimalarea, and percent stenosis in sham treated arteries versus cold treatedarteries. Results Table II contains the data. Mean values shown in thebottom rows indicate that cryotherapy produces less neointimal growthand lower percentage stenosis than observed in sham treated arteries.RESULTS TABLE II Medical Neointimal Lumen % % Medical Injury Animal #Vessel Treatment Area Area Area Stenosis Fracture Score 9108 LAD cryo1.43 2.03 3.42 37.25 0. 0.88 9108 LCX sham 1.25 3.45 2.25 60.53 0. 0.559195 LCX cryo 1.46 5.29 3.32 61.44 15.76 0.56 9195 LAD sham 1.58 6.312.02 75.75 15.47 0.83 9196 LAD cryo 1.57 2.92 3.17 47.95 0. 0.08 9196LCX sham 1.52 2.16 3.61 37.44 0. 0.17 9197 RCA cryo 1.38 5 10 3.82 57.1721.45 0.58 9197 LAD sham 2.00 6.67 3.39 68.30 23.87 0.92 Mean CRYO cryo1.46 3.83 3.43 50. 9.84 0.525 Mean SHAM sham 1.59 4.65 2.82 60. 9.30.617 P-Value 0.45 0.57 0.22 0.29 0.46 0.71

[0129] Experimental IV

[0130] A study was performed to determine if endovascular cryotherapyreduces late neointimal formation in a porcine coronary model ofin-stent restenosis. Normolipemic juvenile swine underwent overstretchballoon angioplasty and stenting. About 4 weeks after this initialangioplasty in-stenting, the in-stent lesions were treated withendovascular cryotherapy or a placebo/sham treatment followed by asecondary stenting. Animals were non-randomly allocated to the activetreatment group (for treatment at a tissue treatment of −5° C.) or theplacebo/control group as outlined in Table III below: TABLE IIITREATMENT GROUPS Volume and Animal Vessel Group TemperatureConcentration 526 LAD I −5° C. 526 LCX II 37° C. 0 526 RCA I −5° C. 527LAD II 7° C. 0 527 RCA I 5° C. 528 LAD I 5° C. 528 RCA II 37° C. 0 529LAD II 37° C. 0 529 LCX I 5° C. 529 RCA I −5° C. 530 LAD I −5° C. 530RCA II 37° C. 0 530 LCX I −5° C. 531 LAD II 37° C. 0 531 RCA I −5° C.

[0131] In-stent lesions were treated with appropriately sized(one-to-one stent to artery ratio) balloon expandable stainless steelstents (Duet™, 18 or 23 mm long, 3.0 to 24.0 mm diameter) to achieve aless than 10% angiographic residual stenosis. Angiographic andintravascular ultrasound analysis were performed after about 28 days,and histological analysis was completed thereafter as describedhereinbelow.

[0132] The animals generally had weights between about 20 and 50 kg, andwere given pre-operative Aspirin (650 mg) and Procardia XL (30 mg) witha small amount of food. After sedation with about 20 mg/kg Ketamine HCLand Xylazine (2 mg/kg), the animals were intubated and mechanicallyventilated with intravenous access established via an ear vein.Adjunctive atropine (0.5 to 1.0 mg IV) was administered as indicated.Continuous anesthesia was maintained with 1% to 2% isoflurane. Sodiumpentobarbital was administered as needed.

[0133] A carotid artery cut-down was performed to gain arterial access.Baseline coronary angiography was performed with an 8F guiding catheterafter the administration of 150 units/kg of intra-arterial Heparin.Additional Heparin was provided so as to maintain the activated clottingtime above 300 seconds.

[0134] Two hundred micrograms of nitroglycerin were administeredintracoronary to prevent vasospasm following overstretch balloonangioplasty. The vessel targeted for treatment was sized by visualestimate using the guiding catheter as a reference. Overstretch ballooninjury was completed using a standard balloon angioplasty catheterhaving a size between about 1.2 and 1.4 times the baseline vesseldiameter.

[0135] Coronary angiography was completed after balloon angioplasty. A3.0 to 4.0 mm diameter stainless steel balloon expanded stent with alength between 13 and 18 mm was implanted at the overstretch site usinga single balloon inflation at 8 to 14 atm. Coronary angiography wascompleted again after stent deployment. The catheters and sheath wereremoved and the carotid artery was treated using standard techniques.The treated animals were allowed to recover from the procedure andreceived 325 mg of Aspirin daily while remaining on a normal diet.

[0136] As outlined in Table III, the treatment vessels included the leftanterior descending artery LAD, the left circumflex artery LCX, and theright coronary artery RCA. The timing of the initial stent injury,cryotherapy or sham treatment, and follow-up are shown in the animal logprovided in FIG. 12A.

[0137] For the endovascular cryotherapy treatment itself baselinecoronary angiography was performed with an 8F guiding catheter afteradministration of intra-arterial Heparin. Activated clotting time wasmaintained at over 300 seconds, with 200 micrograms of nitroglycerinadministered intracoronary. The vessels to be treated were sized by avisual estimate using the guiding catheter as a reference.

[0138] The cryotherapy was administered using a cooled balloon asdescribed hereinabove. A 3.0 to 4.0 mm diameter, 13 to 18 mm longstainless steel balloon expandable stent was implanted at the treatmentsite (within the prior stent) using a single balloon inflation atbetween 8 and 14 atm. Coronary angiography was completed after stentdeployment.

[0139] After completion of stenting, intravascular ultrasound wasperformed by tracking a 30 MHz transducer (CVIS™, from Scimed, of MapleGrove, Minn.) over the guidewire within a 3.2F imaging catheter. Imageswere recorded using a motorized pullback rate of 0.5 mm/sec for off-lineanalysis. This process was completed in an additional artery for theplacebo treatment. Following intravascular ultrasound, the catheters andsheath were removed and the carotid artery treated using standardtechniques. The animals were recovered and monitored, receiving 250 mgof Ticlid for 14 days and 325 mg of Aspirin with a normal diet.

[0140] Each angiogram was evaluated for evidence of intraluminal fillingdefects, side branch occlusion, lumen narrowing, and distal coronaryflow characteristics. The baseline, balloon inflated stent, post injury,and follow-up coronary artery minimal lumen diameters were measured fromnon-overlapped and non-foreshortened views using the guiding catheterfor image calibration, and the data was recorded in mm. The acuteballoon-to-artery ratio (minimal balloon inflated diameter divided bythe baseline lumen diameter) was calculated from this data for eachtreated vessel. The percent stenosis at follow-up, defined as: [(meanreference lumen diameter minus minimal lumen diameter at follow-up/meanreference lumen diameter)]×100, was also calculated.

[0141] Follow-up angiography and intravascular ultrasound were performedat about 28 days after the cryotherapy (or sham treatment) and stentingusing the same techniques described above. The animals were theneuthanized, and the heart was removed immediately with the coronaryarteries pressure perfused with formalin at 60 to 80 mm Hg for one hour.Histological evaluation was then performed.

[0142] Quantitative analysis of the treatment is provided in FIG. 12B.Measurements are generally broken up into measurements for the entiregroup of test animals (Total), measurements for the control vesselswhich received only placebo/sham treatments (Control) and measurementsfor the arteries which received cryotherapy as described above (Cryo).As described, above baseline (BL) measurements were taken beforetreatment, while additional measurements were taken after the initialballoon injury (Balloon). The final two group of measurements were takenafter cryotherapy or the associated placebo/sham (Post Ref), and at thefollow-up date (FU). In addition to the basic reference diametermeasurements, minimum lumen diameters (MLD) and percent diameterstenosis (%DS) are also set forth in FIG. 2B.

[0143] Statistical analysis of the test group was performed usingStatview 4.5 (from Abacus of Berkeley, Calif.), the summary of which isprovided in FIG. 12C. Morphological data are compared by analysis ofvariance (ANOVA) testing. Fischer's protected least significantdifference (PLSD) is shown in FIG. 12C, in which a level of probabilityof statistical significance is indicated by p<0.05 (at which the resultscould be considered to have established statistical significance).

[0144] From the data illustrated in FIGS. 12B and 12C, it can be seenthat the follow-up percent diameter stenosis for the arteries treatedwith cryotherapy is over 17% less than the percent diameter stenosis forthe sham treated arteries in the control group. This represents adecrease in actual or observed stenosis of over 34% when the controlgroup stenosis is used as the baseline.

[0145] While the exemplary embodiments have been described in somedetail by way of example and for clarity of understanding, a variety ofchanges, adaptations, and modifications will be obvious to those ofskill in the art. Hence, the scope of the present invention is limitedsolely by the appended claims.

What is claimed is:
 1. A method for treating hyperplasia or neoplasia ofa blood vessel region, the method comprising: cooling an inner surfaceof the blood vessel region to a temperature and for a time sufficient toremodel the blood vessel such that observed subsequent excessive cellgrowth-induced stenosis of the blood vessel is reduced as compared to astenosis of an equivalently treated uncooled blood vessel region.
 2. Themethod of claim 1, wherein the cooling step effects a relative reductionof the stenosis of at least about 50% of the equivalent vessel regionstenosis.
 3. The method of claim 2, wherein the cooling step inhibits atleast about 8% of the total equivalent vessel region stenosis.
 4. Themethod of claim 2, wherein the cooling time is in a range from about 10to about 30 seconds, and wherein the cooling temperature of the innersurface of the blood vessel is in a range from about 4 to about −31 C.5. A method for inhibiting restenosis of a blood vessel region of amammal, the blood vessel region subjected to a primary treatmenteffecting an initial reduction in stenosis and inducing the restenosis,the method comprising: cooling an inner surface of the blood vesselregion to a temperature and for a time sufficient to remodel the bloodvessel region such that observed restenosis of the blood vessel ismeasurably inhibited.
 6. The method of claim 5, wherein the cooling stepinduces at least one of apoptosis, cell membrane damage, and programmedcell death.
 7. A method for inhibiting restenosis of a blood vesselregion, the blood vessel region subjected to a primary treatmenteffecting an initial reduction in stenosis and inducing the restenosis,the method comprising: cooling an inner surface of the blood vesselregion; reducing cooling so that the inner surface of the blood vesselwarms; re-cooling the warmed inner surface so as to define at least onecooling/warming/cooling cycle, the at least one cycle having coolingtemperatures and times sufficient to remodel the blood vessel regionsuch that the restenosis of the blood vessel is measurably inhibited. 8.A method for treating hyperplasia or neoplasia of a blood vessel region,the method comprising: cooling an inner surface of the blood vesselregion to a temperature and for a time sufficient to induce significantobservable apoptosis of the blood vessel region while avoiding excessivenecrosis of the blood vessel region such that subsequent stenosis of theblood vessel is reduced.
 9. A method for treating hyperplasia orneoplasia of a diseased region of a blood vessel, the method comprising:treating the diseased blood vessel region with a transvascular device,the transvascular device imposing an injury on the vessel, the injurycapable of causing an average injury-induced stenosis; cooling an innersurface of the diseased blood vessel region to a temperature and for atime sufficient to inhibit subsequent excessive cell growth so that theblood vessel undergoes an actual stenosis; and observing that the actualstenosis is less than the average injury-induced stenosis.
 10. Themethod of claim 9, wherein the actual stenosis is at least about 10%less than the average injury-induced stenosis.
 11. The method of claim10, wherein the actual stenosis is at least about 16% less than theaverage injury-induced stenosis.
 12. The method of claim 9, wherein thetreating step comprises deploying a stent within the diseased bloodvessel region.
 13. A method for treating a blood vessel, the bloodvessel having plaque disposed between a lumen and a vessel wall tissue,the method comprising: cooling the vessel wall tissue to a temperaturesufficient to inhibit excessive subsequent cell growth-induced stenosisof the blood vessel, the cooling step performed by: engaging a surfaceof the plaque with a cooling surface; and cooling the plaque with thecooling surface so that the plaque cools the vessel wall tissue.
 14. Themethod of claim 13, wherein the vessel wall tissue is cooled to a targettemperature in a range from about −5° C. to about −15° C.
 15. The methodof claim 14, wherein the cooling surface cools the plaque to atemperature significantly below the target temperature.
 16. The methodof claim 15, wherein the cooling surface cools the plaque to atemperature below the range.
 17. The method of claim 14, wherein thevessel wall tissue is cooled to the target temperature for less thanabout 20 seconds.
 18. The method of claim 17, wherein the vessel walltissue is cooled to the target temperature for a time of at least about10 seconds.
 19. The method of claim 18, wherein a rate of change of thetemperature of the vessel wall tissue is significantly less than a rateof change of a plaque surface temperature.
 20. The method of claim 13,further comprising determining at least one of a cooling surface coolingtemperature and a cooling surface cooling time based at least in part ona thickness of the plaque.